Clonal deletion in the thymus is the mechanism by which T cells recognizing autologous autologous MHC class II antigens, Mls antigens, and the male H-Y antigen are regulated. However, our studies of transgenic mice expressing physiological levels of human insulin indicate that tolerance is not maintained by clonal deletion. These observations support the idea that additional mechanisms operate in the peripheral immune system to maintain unresponsiveness of autoreactive T cells not deleted in the thymus. Using transgenic mice expressing the human insulin gene, we will dissect the mechanisms that maintain peripheral tolerance. To this end, we will determine: 1) whether insulin-specific Th cells are anergic in transgenic mice; 2) the role of Ts cells in tolerance expressed by transgenic mice; 3) whether human insulin-specific CTL can be stimulated in transgenic recipients of nontransgenic T cells; and, 4) if there is evidence for clonal deletion of high avidity T cells in transgenic mice. The second goal of this project is to determine why clonal deletion in the thymus is not effective for circulating autoantigens such as insulin. We hypothesize that clonal deletion of T cells in the thymus is antigen dose-dependent and that the concentration of insulin which reaches the thymus in these transgenic mice is insufficient to cause clonal deletion. A prediction of this hypothesis is that increasing the levels of insulin in the circulation will result in clonal deletion of human insulin-specific T cells. This will be assessed using transgenic mice expressing human insulin under the regulation of the metallothionein promoter or the I-E alpha promoter. The lessons learned from these studies may provide new approaches to the treatment of autoimmune diseases. Thus, our studies will be extended to an immunological disease model of Experimental Allergic Encephalomyelitis (EAE). This system will be used to address the circumstances in which peripheral mechanisms of maintaining self-tolerance fail and whether regulation can be re-instated once the disease occurs. These model systems are particularly relevant for the understanding of autoimune diseases in man in which there is evidence that auto-reactive T cells, present in the peripheral immune system, can escape from the normal regulatory mechanisms. Accumulating evidence suggests that autoimmunity may be involved in the pathogenesis of AIDS. Thus, our studies may be relevant toward understanding the immune dysfunction operating in AIDS and lead to the designing of therapeutic approaches for its treatment.